Micromethods for the biochemical identification of microorganisms have been utilized for many years. For example, several early publications reported the use of reagent-impregnated paper discs and micro-tube methods for differentiating enteric bacteria. Furthermore, interest in miniaturized bacterial identification systems led to the introduction of several commercial systems in the late 1960's. These early miniaturized biochemical identification systems provided advantages such as requiring little storage space, providing extended shelf life, providing standardized quality control, and being relatively easy to use.
The modern broth microdilution test used today has origins in the tube dilution test used as early as 1942 to determine in vitro antimicrobial susceptibility testing (AST) of bacterial isolates from clinical specimens. The broth dilution technique involves exposing bacteria to decreasing concentrations of antimicrobial agents in liquid media by serial two-fold dilution. The lowest concentration of an antimicrobial agent in which no visible bacterial growth occurs is defined as the minimal inhibitory concentration (MIC). The MIC is the standard measure of antimicrobial susceptibility.
The introduction in 1956 of a microtitrator system, using calibrated precision spiral wire loops and droppers for making accurate dilutions rapidly, allowed the development of a serial dilution AST test. The microtitrator system was accurate and allowed the reduction in volumes of antimicrobial agents. The term “microdilution” appeared in 1970 to describe MIC tests performed in volumes of 0.1 mL or less of antimicrobial solution.
Several commercially-available systems automate the microdilution process for MIC/AST testing. For example, the assignees of the various embodiments of the present invention provide a panel-based system (available commercially as the Phoenix™ ID/AST System) capable of performing 100 AST and bacterial identification tests at one time. Such systems include a disposable comprising a sealed and self-inoculating molded polymer tray having 136 microwells containing dried reagents. The tray includes: (1) a bacterial identification (ID) side including dried substrates for bacterial ID; and (2) an AST side having varying concentrations of antimicrobial agents, as well as growth and fluorescent controls at appropriate microwell locations.
In such ID/AST systems, the bacterial ID side utilizes a series of chromogenic and fluorogenic biochemical tests to determine the identification of a bacterial organism. Both growth-based and enzymatic substrates are employed to cover different types of reactivity within the range of taxa that may be present in a given sample. These ID tests are based on microbial utilization and subsequent degradation of substrates detected by various indicator systems. Acid production is indicated by a change in phenol red indicator when an isolate is able to utilize a carbohydrate substrate. Furthermore, chromogenic substrates produce a yellow color upon enzymatic hydrolosis of either p-nitrophenyl or p-nitroanilide compounds. Enzymatic hydrolosis of fluoregenic substrates results in the release of a fluorescent coumarin derivative. Bacterial organisms that utilize a specific carbon source reduce the resazurin-based indicator. In addition, other tests are provided on the bacterial ID side to detect the ability of a bacterial organism to hydrolyze, degrade, reduce, or otherwise utilize a given substrate present in the microwells of the bacterial ID side.
Furthermore, the AST side of panel-based systems utilizes broth-based microdilution. For example, the Phoenix™ system utilizes a redox indicator for the detection of organism growth in the presence of a given antimicrobial agent. Continuous measurements of changes to the indicator, as well as bacterial turbidity measurements (as described further herein) may be used in the determination of bacterial growth. Each AST panel configuration contains several antimicrobial agents with a wide range of two-fold doubling dilution concentrations. Organism ID is used in the interpretation of MIC values of each antimicrobial agent.
Such panel-based systems are conventionally provided as a disposable component of an overall ID/AST system (such as the Phoenix™ system, for example). In such systems, the disposable panels must be exposed to a sample having a selected organism density (defined, for example, by the turbidity of the sample relative to the McFarland (McF) scale). For example, the Phoenix™ system often utilizes panels that have been inoculated with a targeted organism density of either 0.25 McF or 0.5 McF.
Thus, the effective use of ID/AST systems requires the manual preparation of a panel inoculum having a selected concentration of particles (expressed as a turbidity, for example) that is standardized relative to the McFarland scale. Therefore, improvements in inoculum preparation and handling are desirable.